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STRUCTURE BASED DESIGN of PEPTIDOMIMETIC INHIBITORS of the MLL1-WDR5 INTERACTION By STRUCTURE BASED DESIGN OF PEPTIDOMIMETIC INHIBITORS OF THE MLL1-WDR5 INTERACTION by Hacer Karatas A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Medicinal Chemistry) in The University of Michigan 2012 Doctoral Committee: Professor Shaomeng Wang, Chair Professor David H. Sherman Associate Professor Anna K. Mapp Assistant Professor Yali Dou Assistant Professor Sylvie Garneau-Tsodikova © Hacer Karatas 2012 To my parents Sultan and Ebubekir Karatas. ii Acknowledgements First and foremost, I would like to thank to my advisor Dr. Shaomeng Wang for his guidance and support throughout my doctoral study. I am deeply grateful to Dr. Yali Dou and Dou lab members for their extensive and constructive collaboration. I would like to thank Elizabeth C. Townsend for her work in Biacore assay, immunoprecipitation studies, HMTase assay and preparing WDR5 for the binding assay. I also like to thank Dr. Fang Cao who performed qRT-PCR and cell growth inhibition assay. I am very grateful to Dr. Yong Chen for the crystal structure studies of MM-101 and MM-102. I would like to thank Dr. Denzil Bernard for modeling and MD simulation studies. I also like to thank Dr. Liu Liu for the PAMPA permeability assay. I’m grateful to Dr. Zaneta Nikolovska-Coleska for her training regarding the development of FP- based binding assay. I would like to acknowledge my committee members Dr. David Sherman, Dr. Anna K. Mapp, Dr. Yali Dou and Dr. Sylvie Garneau-Tsodikova for their helpful discussion and feedback throughout my studies. I have had the opportunity of working with a number of outstanding researchers in the Wang Lab. I would like to thank all the past and current members, especially Dr. Jianyong Chen, Dr. Angelo Aguilar, Yujun Zhao, Haibin Zhou and Shanghai Yu for the constructive discussions and their invaluable feedback. iii I would like to give my special thanks to Dr. George W. A. Milne for his critical reading of this dissertation. I’m deeply grateful to my former advisor Dr. Hakan Goker, former mentors Dr. Sureyya Olgen and Ilhan Oz for their guidance and training. My heartfelt thanks go to my little family here in Ann Arbor, especially Arzucan Ozgur, Hande Kocak, Thuy Vu, Ayse and Guniz Buyuktur, Bilgen Ekim and Artagun Yesildere. Lastly but mostly, my gratitude is to my family whose support made this work possible. I’m grateful to my parents Ebubekir and Sultan Karatas, my brother Mehmet Karatas, my sister Esma K. Karatas and my dear niece Zeynep Tabak who never stopped dreaming with me. New members of the family, Meral and Duru Karatas, will make our dreams even bigger. iv Table of Contents Dedication………………………………………………………………………………....ii Acknowledgements ............................................................................................................ iii List of Figures .................................................................................................................. viii List of Tables ...................................................................................................................... x List of Schemes ................................................................................................................. xii List of Appendices ........................................................................................................... xiii List of Abbreviations ....................................................................................................... xiv Abstract ............................................................................................................................ xvi CHAPTER 1 ....................................................................................................................... 1 Introduction ..................................................................................................................... 1 1.1 Histone Modifications ...................................................................................... 2 1.2 Histone Lysine Methylation ............................................................................. 3 1.3 H3K4 Methylation ............................................................................................ 4 1.4 Organization and Function of the MLL1 Complex .......................................... 5 1.5 Leukemic Transformation with MLL1 Fusion Proteins ................................... 7 1.6 Targeting the MLL1-WDR5 Interaction .......................................................... 9 CHAPTER 2 ..................................................................................................................... 12 Development of FP-Based Binding Assay ................................................................... 12 2.1 Design and Optimization of Fluorescently Tagged Peptides for Assay Development ................................................................................................... 13 2.2 Development and Optimization of Competitive Binding Assay Conditions . 14 CHAPTER 3 ..................................................................................................................... 16 Analysis of the MLL1 and Histone 3 (H3) Peptides Binding to WDR5 ...................... 16 3.1 Determination of the Minimal Motif in MLL1 for High-Affinity Binding to WDR5 ............................................................................................................. 16 v 3.2 Role of Two Intramolecular Hydrogen Bonds in MLL1 Peptides for High- Affinity Binding to WDR5 ............................................................................. 18 3.3 Binding of H3 Peptides to WDR5 .................................................................. 22 3.4 Conclusion ...................................................................................................... 24 CHAPTER 4 ..................................................................................................................... 26 SAR Studies of Ligand-WDR5 Interaction .................................................................. 26 4.1 Determination of Favorable Residues for Binding to WDR5 at Ala1, Arg2 and Ala3 Positions in Ac-ARA-NH2 ..................................................................... 26 4.2 Further Modifications Using Constrained Hydrophobic Side Chains to Target S1 and S4 Pockets ........................................................................................... 32 4.3 Evaluation of Permeability of Peptides and Peptidomimetics ........................ 36 4.4 Further Modifications at the N- and C- terminus to Improve Cell Permeability ........................................................................................................................ 36 4.5 Design of Potent and Cell Permeable Peptidomimetics (MM-101 and MM- 102) ................................................................................................................. 39 4.6 Determination of Crystal Structures of MM-101 and MM-102 in Complex with WDR5 ..................................................................................................... 40 4.7 Conclusion ...................................................................................................... 43 CHAPTER 5 ..................................................................................................................... 45 Biochemical and Biological Evaluation MLL1-WDR5 Interaction Inhibitors ............. 45 5.1 Solubility Analysis of MM-101 ...................................................................... 46 5.2 Stability Analysis of MM-101 against Trypsin Degradation ......................... 46 5.3 Stability Analysis of MM-101 in Cell Culture Media .................................... 48 5.4 Inhibition of MLL1-WDR5 Interaction .......................................................... 49 5.5 Inhibition of H3K4 Methyltransferase Activity of the MLL1 Core Complex 50 5.6 Inhibition of MLL1 Target Gene Expression ................................................. 53 5.7 Inhibition of Cell Growth ............................................................................... 54 5.8 Conclusion ...................................................................................................... 55 CHAPTER 6 ..................................................................................................................... 58 Summary and Future Directions ................................................................................... 58 6.1 Rationale and Design of the Study ................................................................. 58 6.2 Significance of the Study ................................................................................ 60 6.3 Future Directions ............................................................................................ 61 Appendices ........................................................................................................................ 65 vi Appendix A ....................................................................................................................... 66 Experimental ................................................................................................................. 66 Appendix B ....................................................................................................................... 79 Spectral Data ................................................................................................................. 79 Bibliography ....................................................................................................................
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